Refolding Record:
| Protein | |
|---|---|
| Protein Name | Phytase |
| Abbreviated Name | phy |
| SCOP Family | Thermostable phytase (3-phytase) |
| Structure Notes | |
| Organism | Bacillus subtilis |
| UniProt Accession | B1GSN6 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 383 |
| Molecular Weight | 41863.5 |
| Pi | 5.08 |
| Molecular Weight | 41863.5 |
| Disulphides | Unknown |
| Full Sequence |
MNHSKTLLLTAAAGLMLTCGAVSSQAKHKLSDPYHFTVNAAAETEPVDTAGDAADDPAIWLDPKTPQNSKLITTNKKSGLVVYSLDGKMLHSYNTGKLNNVDIRYDFPLNGKKVDIAAASNRSEGKNTIEIYAIDGKNGTLQSMTDPDHPIATAINEVYGFTLYHSQKTGKYYAMVTGKEGEFEQYELKADKNGYISGKKVRAFKMNSQTEGMAADDEYGRLYIAEEDEAIWKFSAEPDGGSNGTVIDRADGRHLTPDIEGLTIYYAADGKGYLMASSQGNSSYAIYDRQGKNKYVADFRITDGPETDGTSDTDGIDVLGFGLGPEYPFGIFVAQDGENIDHGQKANQNFKIVPWERIADQIGFRPLANEQVDPRKLTDRSGK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Rao DE, Rao KV, Reddy VD. (2008) J Appl Microbiol, 1, 1 |
| Project Aim | Cloning and expression |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3-6 h |
| Expression Vector | pET21a |
| Expression Protocol | Hundred millilitres of Luria Bertani (LB) medium containing 70 μg ml−1 ampicillin was inoculated with 1 ml each of overnight culture of the different clones and agitated at 220 rev min−1 at 37°C. The cultures were induced with 1 mmol l−1 isopropyl β-d-thiogalactopyranoside (IPTG) once the OD600 reached 0·8 (log phase), and were allowed to grow under the same conditions. The uninduced and 3, 6 h and overnight induction samples were collected. Cells were lysed with 2× SDS buffer (Sambrook and Russel 2001) and electrophoresed on a 12% SDS-PAGE gel along with a standard protein marker. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.8 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mmol l−1 Tris-Cl, pH 8·0, 100 mmol l−1 NaCl) containing 0·5% Triton X-100 |
| Solubilization Buffer | 5 ml 8·0 mol l−1 urea in Tris-Cl (pH 7·0) buffer |
| Refolding Buffer | 100 mmol l−1 Tris-Cl, pH 7·0, 0·5 mmol l−1 CaCl2) containing one of the different refolding co-solutes, viz 20% sucrose, 0·4 mol l−1l-arginine, 2·0 mol l−1 proline, 20% glycerol and 50 mmol l−1 glycine |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 7.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The denatured protein was diluted to a final concentration of 0·1 mg ml−1 in refolding buffer (100 mmol l−1 Tris-Cl, pH 7·0, 0·5 mmol l−1 CaCl2) containing one of the different refolding co-solutes, viz 20% sucrose, 0·4 mol l−1l-arginine, 2·0 mol l−1 proline, 20% glycerol and 50 mmol l−1 glycine. Protein aggregation was monitored by the percentage transmittance of these solutions at OD600. The purified and denatured protein was diluted to a final concentration of 0·1 mg ml−1 in refolding buffer (100 mmol l−1 Tris-Cl, pH 7·0, 0·5 mmol l−1 CaCl2) with and without proline. Experiments were carried out at 10°C and room temperature (28°C) to optimize the appropriate protein refolding temperature. Enzyme activity was tested every 24 h for a period of 1 week.To optimize the appropriate concentration of proline for protein refolding, the refolding experiments were carried out using 0, 0·5, 1·0, 1·5, 2·0 and 2·5 mol l−1 proline in the refolding buffer. |
| Refolding Assay | Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 20% See note |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | The refolding buffer is containing one of the different refolding co-solutes, viz 20% sucrose, 0·4 mol l−1l-arginine, 2·0 mol l−1 proline, 20% glycerol and 50 mmol l−1 glycine |